Modulation of the immune system is desirous in a variety of contexts, from inhibiting an autoimmune response, to controlling infectious disease and inhibiting graft/tissue rejection. The principal approach to mitigate rejection is the pharmacological suppression of the immune system of the recipient. With this in mind, most immunomodulatory compounds that are currently utilized are immunosuppressive. Since the early 1960's the availability of these immunosuppressive agents have been restricted to only a few drugs. However, in the early 1980's in addition to azathioprine and corticosteroids, cyclosporine became widely available and has been the drug of choice ever since. (Kobashigawa, Trans. Proc. 30:1095–1097, 1998; Isoniemi, Ann. Chi. Gyn. 86:164–170, 1997). However, the newer immunosuppressive agents are relatively few in number and also suffer from many of the undesirable side-effects associated with earlier agents. While these drugs have been used to increase survival times for transplanted organs, either as single agents or in combination with other immunosuppressants, many are also useful for treating inflammatory and autoimmune diseases, delayed hypersensitivity, graft versus host diseases and similar immune system associated diseases.
Currently used immunosuppressive drugs include antiproliferative agents, such as methotrexate, azathioprine, and cyclophosphamide. Since these drugs affect mitosis and cell division, they have severe toxic effects on normal cells with high turn-over rate such as bone marrow cells and the gastrointestinal tract lining. (Miller, Semin. Vet. Med. Surg. 12(3):144–149, 1997) Accordingly, marrow depression and liver damage are common side effects.
Antiinflammatory compounds used to induce immunosuppression include adrenal corticosteroids such as dexamethasone and prednisolone. The common side effects observed with the use of these compounds are frequent infections, abnormal metabolism, hypertension, and diabetes.
Other immunosuppressive compounds currently used to inhibit lymphocyte activation and subsequent proliferation include cyclosporine, FK506, and rapamycin. These drugs inhibit either the calcium dependent phosphatase calcineurin (cyclosporine and FK506) or inhibit the p70S6 kinase that is important for growth factor induced signaling (rapamycin). (Liu et al., Cell 66:807–815, 1991; Henderson et al., Immun. 73:316–321, 1991; Bierer et al., Curr. Opin. Immun. 5:763–773, 1993; Isoniemi (supra)). Cyclosporine and its relatives are among the most commonly used immunosuppressants. Cyclosporine is typically used for preventing or treating organ rejection in kidney, liver, heart, pancreas, bone-marrow, and heart-lung transplants, as well as for the treatment of autoimmune and inflammatory diseases such as Crohn's disease, aplastic anemia, multiple-sclerosis, myasthenia gravis, uveitis, biliary cirrhosis, etc. However, cyclosporines suffer from a small therapeutic dose window and severe toxic effects including nephrotoxicity, hepatotoxicity, hypertension, hirsutism, cancer, and neurotoxicity. (Philip and Gerson, Clin. Lab. Med. 18(4):755–765, 1998; Hojo et al., Nature 397:530–534, 1999).
Additionally, monoclonal antibodies, such as OKT3 have been used to prevent and/or treat graft rejection. Introduction of monoclonal antibodies into a patient, as with many biological materials, induces several side-effects, such as rigors and dyspnea. (Richards et al., Cancer Res. 59(9):2096–2101, 1999).
Within the context of many life-threatening diseases, organ transplantation is considered a standard treatment and, in many cases, the only alternative to death. The immune response to foreign cell surface antigens on the graft, encoded by the major histocompatibility complex (MHC) and present on all cells, generally precludes successful transplantation of tissues and organs unless the transplant tissues come from a compatible donor and the normal immune response is suppressed. Other than identical twins, the best compatibility and thus, long term rates of engraftment, are achieved using MHC identical sibling donors or MHC identical unrelated cadaver donors (Strom, Clin. Asp. Autoimm. 4:8–19, 1990). However, such ideal matches are difficult to achieve. Further, with the increasing need of donor organs an increasing shortage of transplanted organs currently exists. Accordingly, xenotransplantation has emerged as an area of intensive study, but faces many hurdles with regard to rejection within the recipient animal (Kaufman et al., Annu. Rev. Immunol. 13:339–367, 1995).
The host response to an organ allograft involves a complex series of cellular interactions among T and B lymphocytes as well as macrophages or dendritic cells that recognize and are activated by foreign antigen (Strom, supra; Cellular and Molecular Immunology, Abbas et al. (Eds.), W B Saunders Co., Penn., 1994). Co-stimulatory factors, primarily cytokines, and specific cell—cell interactions, provided by activated accessory cells such as macrophages or dendritic cells are essential for T-cell proliferation. These macrophages and dendritic cells either directly adhere to T-cells through specific adhesion proteins or secrete cytokines that stimulate T-cells, such as IL-12 and IL-15 (Strom, In: Organ Transplantation:Current Clinical and Immunological Concepts, 1989). Accessory cell-derived co-stimulatory signals stimulate activation of interleukin-2 (IL-2) gene transcription and expression of high affinity IL-2 receptors in T-cells (Pankewycz et al., Transplantation 47:318, 1989; Cantrell et al., Science 224:1312, 1991; Williams et al., J. Immunol. 132:2330–2337, 1984). IL-2, a 15 kDa protein, is secreted by T lymphocytes upon antigen stimulation and is required for normal immune responsiveness. IL-2 stimulates lymphoid cells to proliferate and differentiate by binding to IL-2 specific cell surface receptors (IL-2R). IL-2 also initiates helper T-cell activation of cytotoxic T-cells and stimulates secretion of interferon-γ (IFN-γ) which in turn activates cytodestructive properties of macrophages (Farrar et al., J. Immunol. 126:1120–1125, 1981). Furthermore, IFN-γ and IL-4 are also important activators of MHC class II expression in the transplanted organ, thereby further expanding the rejection cascade by enhancing the immunogenicity of the grafted organ (Pober et al., J. Exp. Med., 157:1339, 1983; Kelley et al., J. Immunol., 132:240–245, 1984).
The current model of a T-cell mediated response suggests that T-cells are primed in the T-cell zone of secondary lymphoid organs, primarily by dendritic cells. The initial interaction requires cell to cell contact between antigen-loaded MHC molecules on antigen-presenting cells (APCs) and the T-cell receptor (TCR)/CD3 complex on T-cells. Engagement of the TCR/CD3 complex induces CD154 expression predominantly on CD4 T-cells that in turn activate the APC through CD40 engagement, leading to improved antigen presentation (Grewal et al., Ann. Rev Immunol. 16:111–135, 1998). This is caused partly by upregulation of CD80 and CD86 expression on the APC, both of which are ligands for the important CD28 costimulatory molecule on T-cells. However, engagement of CD40 also leads to prolonged surface expression of MHC-antigen complexes, expression of ligands for 4-1 BB and OX-40 (potent costimulatory molecules expressed on activated T-cells). Furthermore, CD40 engagement leads to secretion of various cytokines (e.g., IL-12, IL-15, TNF-α, IL-1, IL-6, and IL-8) and chemokines (e.g., Rantes, MIP-1α, and MCP-1), all of which have important effects on both APC and T-cell activation and maturation (Mackey et al., J. Leukoc. Biol. 63:418–428, 1998).
Similar mechanisms are involved in the development of autoimmune disease, such as type I diabetes. In humans and non-obese diabetic mice (NOD), insulin-dependent diabetes mellitus (IDDM) results from a spontaneous T-cell dependent autoimmune destruction of insulin-producing pancreatic β cells that intensifies with age. The process is preceded by infiltration of the islets with mononuclear cells (insulitis), primarily composed of T lymphocytes (Bottazzo et al., J. Engl. J. Med., 113:353, 1985; Miyazaki et al., Clin. Exp. Immunol., 60:622, 1985). A delicate balance between autoaggressive T-cells and suppressor-type immune phenomena determine whether expression of autoimmunity is limited to insulitis or progresses to IDDM. In NOD mice, a model of human IDDM, therapeutic strategies that target T-cells have been successful in preventing IDDM (Makino et al., Exp. Anim., 29:1, 1980). These include neonatal thymectomy, administration of cyclosporine, and infusion of anti-pan T-cell, anti-CD4, or anti-CD25 (IL-2R) monoclonal antibodies (mAbs) (Tarui et al., Insulitis and Type I Diabetes. Lessons from the NOD Mouse, Academic Press, Tokyo, p. 143, 1986). Other models include those typically utilized for autoimmune and inflammatory disease, such as multiple sclerosis (EAE model), rheumatoid arthritis, graft versus host disease, systemic lupus erythematosus (systemic autoimmunity—NZBxNZWF1 model), and the like. (see, for example, Theofilopoulos and Dixon, Adv. Immunol. 37:269–389, 1985; Eisenberg et al., J. Immunol. 125:1032–1036, 1980; Bonneville et al., Nature 344:163–165, 1990; Dent et al., Nature 343:714–719, 1990; Todd et al., Nature 351:542–547, 1991; Watanabe et al., Biochem Genet. 29:325–335, 1991; Morris et al., Clin. Immunol. Immunopathol. 57:263–273, 1990; Takahashi et al., Cell 76:969–976, 1994; Current Protocols in Immunology, Richard Coico (Ed.), John Wiley & Sons, Inc., Chapter 15, 1998).
The aim of all rejection prevention and autoimmunity reversal strategies is to suppress the patient's immune reactivity to the antigenic tissue or agent, with a minimum of morbidity and mortality. Accordingly, a number of drugs are currently being used or investigated for their immunosuppressive properties. As discussed above, the most commonly used immunosuppressant is cyclosporine, but usage of cyclosporine has numerous side effects. Accordingly, in view of the relatively few choices for agents effective at immunosuppression with low toxicity profiles and manageable side effects, there exists a need in the art for identification of alternate immunosuppressive agents. The present invention meets this need and provides other related advantages.